Throughout this application, various articles and books are referenced by authorship and date. The full bibliographic citation for each publication can be found at the end of the specification, immediately preceding the claims. In addition, certain terms used in this application are defined in the definitions section immediately following the references.
The present invention relates to the discovery of a novel endohpytic fungus, Muscodor albus (M. albus), that makes a series of volatile compounds that are inhibitory and/or lethal to other microbes including those found in human wastes such as Escherichia coli (E. coli) and Vibrio cholerae. In a preferred embodiment, the gas phase of these novel fungi, including M. albus and fungi related to it such as Muscodor roseus (M. roseus), are used in the treatment of human wastes, and in particular, those contained in the WAG BAG™ (defined below). The volatile gases of M. albus consist of at least 28 compounds, most of which have been identified. An artificial mixture of these compounds, in the gas phase, largely mimics the effects of the fungal gases relative to their bioactivity (Strobel et al., 2001). The present invention also relates to the preparation, growth, formulation and storage of the M. albus so that it can ultimately placed into the WAG BAG™ or similar bags used in connection with portable toilets and effectively be wetted by human urine and begin to make its gases and inhibit and kill harmful microorganisms found in human wastes.
This invention addresses several extremely important developments that relate to modern concerns and needs for the safe and effective treatment of liquid and solid human wastes. This need arises especially with respect to people who are removed, from one reason or another, from sanitary facilities. This may include those who are serving in defense forces in the field or in a recreational mode. It may include people who are in emergency or natural disaster situations, or in conditions of national war or other political disasters. In each of these situations, in order to survive, the human body needs to effectively pass liquid and solid wastes. Unfortunately, in many cases, both environmental as well health hazards are caused when these wastes are not properly disposed (FIG. 1). It is for these reasons that Phillips Environmental Products, Inc. has developed a biodegradable WAG BAG™ for use in connection with the Portable Environmental Toilet (PETT®) (U.S. Pat. No. 6,341,386). The WAG BAG™ contains an agent that absorbs and gels liquid, and upon burial or proper disposal, will degrade into harmless organic material. What is needed is a method to immediately treat the wastes so that in the first ten days to two weeks after collection in the bag there is an effective reduction of the majority of harmful bacterial organisms, followed by degradation of the dead bacteria and unprocessed polymers such as cellulose, hemicellulose and lignins contained in the human solid waste. The application of M. albus in a form that can be placed in the WAG BAG™ is also critical to the treatment processes. Data supporting this treatment process via the use of M. albus are included in this patent application.
M. albus was isolated as an endophytic fungus growing within the limb tissues of Cinnamomum zeylanicum in the Caribbean rainforest of Honduras in 1997 (Strobel et al., 2001). Endophytes have been classified as microbes living within the living tissues of host plants without causing any overt symptoms of disease or other harm (Bacon and White, 2000). M. albus produced no spores in culture, and thus it had to be classified primarily according to its molecular biological characteristics (Woropong et al., 2001). This endophytic microbe is of the xylariaceae family. It has a close relative, M. roseus, which also makes volatile antibiotics (Woropong et al., 2002). When M. albus was originally isolated, it was noted that it killed all other microbes that were also emerging from the cinnamon tree limbs that were in culture. This was a significant observation because it showed that the fungus was making gaseous compounds that were lethal to other microbes, in other words, volatile antimicrobials. This observation appears to be the first time that anyone has noticed such a phenomenon from an endophytic microbe, although others have noted the fact that fungi produce odors, but not with lethal microbial effects (Bjurman and Kristensson, 1992; Dennis and Webster, 1971; Rapior et al., 2000 and Schnurer et al., 1999).
Thus it was necessary to trap and identify the individual compounds produced by this fungus and then mimic the effects in a similar manner. It was also necessary to devise a bioassay test and to determine the microbes and other organisms that may be sensitive to the effect of these fungal gaseous compounds. It was learned that the gases are lethal or inhibitory to many human pathogenic fungi and bacteria, including, but not limited to, E. coli, Vibrio cholerea, Candida albicans and Aspergillus fumigatus. The former two bacteria are found in human wastes and are pathogenic to humans. Usually, only a 24-hour exposure to the fungal gases is required to produce lethal or inhibitory effects. In some instances, however, a longer exposure is required.
This invention also relates to the discovery of several other gas-producing fungi that are also effective in killing other microbes. They were isolated by using M. albus, as the source of selective gases, in the presence of plant material suspected of supporting other gas-producing endophytes. It turns out that other fungal relatives in the xylariaceae family are not sensitive to the M. albus gases. Thus, the volatile antibiotics selectively eliminate all microbes except its relatives and other volatile antimicrobial producers. At least two other volatile antimicrobial endophytes have been isolated, and they too are effective in killing other microbes. These fungi have been designated as A3-5 and A10-1 (Woropong et al., 2002). They resemble M. albus in that they produce antimicrobial volatiles and do not make spores, but the antimicrobial volatiles they produce are not identical to those of M. albus. These fungi are called M. roseus, and they are as effective as M. albus in controlling other microbes (Worapong et al., 2002).